When working with primary cells, it is important to remember that they are not cell lines and cannot be treated the same. Because ScienCell has extensively worked with primary cells, we are very familiar with the common problems researchers encounter when culturing them. Here are six common mistakes that are made with primary cells and how to correct these mistakes.
Mistake #1: Thawing a vial of primary cells in a water bath for an extended period of time
Correction #1: Primary cells are very sensitive to the thawing process so it is important that the vial be placed in a 37oC water bath, held and rotated gently until the contents are just thawed. Then remove the vial from the water bath promptly and transfer in into a sterile hood. Make sure your culture vessel is ready before thawing so the cells can immediately be seeded and placed in the incubator.
Mistake #2: Centrifuging primary cells directly after thawing a vial
Correction #2: We do not recommend centrifuging the cells after thawing because the centrifugation procedure is more harmful than the minimal DMSO residue. Remember to change medium the following day after reviving primary cells to remove any residual DMSO.
Mistake #3: Allowing primary cells to become too confluent
Correction #3: Primary cells can become senescent when grown to 100% confluent. Remember that primary cells are not 100% pure, so it is important to minimize growth of contaminating cells. We recommend subculturing primary cells when they are 90-95% confluent.
Mistake #4: Over-trypsinization when passaging primary cells
Correction #4: When passaging cells, use low concentrations of trypsin and monitor cells closely under the microscope. Also, remember to completely neutralize the cells after trypsinization because any active trypsin will damage the cells. We specifically recommend a trypsin neutralization solution (Cat #0113) specially formulated for primary cells, but 10% serum or soybean trypsin inhibitor (Cat #0173) can also be used.
Mistake #5: Primary cells can easily be refrozen
Correction #5: Normally we do not recommend refreezing primary cells as this can promote a senescent phenotype and/or cause functional changes. Primary cells are extremely sensitive and refreezing may result in cell death or damage.
Mistake #6: Primary cells can proliferate indefinitely
Correction #6: Unlike cell lines, primary cells have a limited expansion capacity. We recommend using primary cells as early as possible for experiments to prevent genetic drift. In addition, if you are working with a difficult cell type you should monitor cell morphology closely for contaminating cells as they can take over the culture over time.
It is very important to keep these six technical tips in mind when you are culturing primary cells. Ultimately, if you treat your cells carefully they will perform better for your experiments.
If you have any further technical questions regarding primary cell culture please contact us at firstname.lastname@example.org.
As to mistake 1, I use a different technique that works very well, especially when DMSO or other cryopreservatives are used, as it simultaneously allows fast thawing and dilution of the cryopreservative. Instead of placing the vial in the water bath, I add a small volume of warm culture medium to the cryovial and after pippetting up and down, I transfer it to a culture flask. The process is repeated until all the content in the vial is thawed and transferred.
Thanks for the comment! There are a variety of ways to thaw cells, however, we recommend thawing the primary cells in a water bath to minimize handling of the cells. This is very important because primary cells are extremely sensitive especially after thawing the cells. Extra pipetting could damage the cells.
Thanks. Very helpful even though I work with cell lines
Thanks for the comment! We’re glad you find it helpful.
What is the best way to freeze and thaw primary rat neonatal skeletal muscle
Thanks for the comment! We do not recommend refreezing the primary cells as this can cause functional and morphological changes in the cells.